LncRNA Research

Functional LncRNA PCR Panel: From the unknown to biological significance

Long non-coding RNAs (LncRNAs) are a class of non-protein coding RNAs longer than 200 nt. LncRNAs can regulate gene expression by various mechanisms with biological functions in, for example, embryonic development [1], stem cell pluripotency, cell lineage specification, genomic imprinting, cardiac development [2], hematopoiesis, immunity [3], and endocrine systems [4]. Dysregulated expression of lncRNAs has been associated with diseases ranging from cancer [6], neurodegenerative, cardiovascular [5], kidney, and diabetic diseases. Profiling LncRNA expression is key to the understanding of their functions and molecular mechanisms. LncRNA expression is much higher in specificity than mRNA, which is explored as a new class of biomarkers.

Although the majority of lncRNAs are still functionally unknown, intense lncRNA research has identified and accumulated functional information for many lncRNAs. The Functional LncRNA PCR Panel is a highly sensitive and accurate tool to study these lncRNAs in your biology and disease systems, as well as biomarker applications. The lncRNA classification, mechanisms of action, biological functions, disease associations, and clinical uses are annotated and analyzed with the panel.

Classification of LncRNAs

Based on their genome positions relative to the neighboring protein-coding genes, lncRNAs can be categorized as antisense, enhancer, intergenic, bidirectional and intronic lncRNAs (Figure 1), which are strongly correlated with the mechanisms of how they regulate the target genes. For example, enhancer lncRNAs mediate short- and long-range interactions between the enhancers from which they are transcribed and the target regulatory elements such as promoters. The nascent lncRNA transcripts often tether chromatin modifiers to epigenetically control neighboring genes. Also, the act of lncRNA transcription itself can generate changes in chromatin accessibility or protein binding, independent of its lncRNA products.

Figure 1 | Classification of lncRNAs based on genomic locations. Antisense lncRNAs are transcribed from the antisense strand and overlap with the protein-coding gene sequence. Enhancer lncRNAs are located in enhancer regions. Intergenic lncRNAs are transcripts located from protein-coding genes at distances of more than 1~5 kb. Bidirectional lncRNAs are transcribed within 1 kb of promoters of the protein-coding transcript in the opposite direction. Intronic lncRNAs are derived from the intron of a coding gene.

Mechanisms of action

LncRNAs act through diverse mechanisms that rely on their sequence, secondary or tertiary structures. The subcellular localization of lncRNAs in the nuclear, cytosolic, or both compartments provides a strong indication of the molecular mechanism. Nuclear lncRNAs have the access to chromatin and genomic DNA, where they can act as molecular scaffolds, aid alternative splicing, or modify chromatin structures. Cytosolic lncRNAs more likely regulate the targets in trans at post-transcriptional levels, promote or inhibit mRNA degradation by acting as, for example, miRNA sponges, and modulate translation (Figure 2).

Figure 2 | Mechanisms of lncRNA action [4]. Most lncRNAs are nuclear and their most common mechanism of action is via recruitment of chromatin modifiers to DNA. These chromatin modifiers can be repressive, activating (such as transcriptional mediators) or other modifiers such as hnRNPs as nuclear organization factors. Some lncRNAs bind to specific proteins and act as scaffolds within ribonucleoprotein complexes. In the cytosol, lncRNAs can act at the post-transcriptional level as sponges for miRNAs, therefore inhibiting the actions of miRNAs on mRNAs. hnRNP, heterogeneous ribonucleoprotein; PRC, polycomb repressor complex.

Genomic imprinting is an important developmental mechanism [7]. For instance, lncRNA Airn is essential for the silencing of the Igf2r/Slc22a2/Slc22a3 gene cluster on the paternal chromosome. The antisense lncRNA Kcnq1ot1 regulates the silenc¬ing function of the imprinting control region of Kcnq1 on the unmethylated paternal chromosome. During development in females, the lncRNA Xist initiates the inactivation of one of the entire X chromosomes, while leaving the other X chromosome active.

Stem cell differentiation

More than 100 lncRNA gene promoters are bound by stem cell factors. Disruption of these lncRNAs can alter cell differentiation [8]. One of them, lincRNA-RoR, is involved in the reprogramming of fibroblasts back to a pluripotent state, illustrating the important roles of lncRNAs in both normal development and the maintenance of adult stem cell pools.

Embryonic development

HOX family of transcription factors specify cell differentiation and regulate the embryo body plan [3]. The HOX clusters of genes are regulated by lncRNA HOTTIP that binds WDR5, a key component of histone-modifying MLL1 complex, to catalyze activating H3K4me3 marks and maintain gene activation in the HOXA locus. Another lncRNA HOTAIR acts as a repressor of the HOXD cluster by recruiting repressive complex PRC2.

Lipid metabolism and adipogenesis

Certain LncRNAs are found to control lipid metabolism, influence lipid homeostasis, and regulate adipogenesis in liver [9]. APOA1 is a major component of high-density lipoprotein (HDL). APOA1-AS, antisense transcript of APOA1, negatively regulates APOA1 expression in vitro and in vivo. Another lncRNA, NEAT1, regulates PPARγ2 splicing during adipogenesis and also mediates miR-140 induced adipogenesis.

Hematopoiesis and Immunity

Many lncRNAs participate in different stages of immune system development and activation [3]. For example, Lnc-DC controls dendritic cell differentiation by promoting phosphorylation and nuclear translocation of a key DC transcription factor STAT3. LncRNA PACER is upregulated after LPS stimulation in human macrophages and selectively regulates the expression of its neighboring gene COX-2. Furthermore, lncRNA THRIL, is essential for basal and inducible expression of proinflammatory cytokine TNF, through interaction with an hnRNP.

Cardiac development and function

Many lncRNAs are differentially expressed during cardiac development, several of which have been characterized function roles [2]. For example, depletion of lncRNA BVHT severely impairs embryonic stem cells to produce differentiated cardiomyocytes. ENDRR, an lncRNA exclusively expressed in cardiac mesoderm progenitors, is involved in cardiac lineage commitment and lateral mesoderm differentiation to gives rise to the ventral body wall and heart.

Association with human diseases

The number of lncRNAs known to be involved in diseases is increasing at a rapid rate. However, more efforts are needed to elucidate the lncRNA functions in diseases and to explore their therapeutic and biomarker potentials.

Cancer

LncRNAs drive almost every cancer hallmark including growth, proliferation, metastasis and survival [6] (Figure 3). For instance, in T cell acute lymphoblastic leukemia, lncRNA LUNAR1 is induced by oncogene Notch1 to upregulate insulin-like growth factor 1 receptor expression to promote cell growth. Androgen signaling in prostate cancer also relies on a number of lncRNAs such as PCGEM1, PRNCR1, and HOTAIR that directly interact with androgen receptor, or inhibit the repressors of androgen receptor (CTBP1-AS).

Figure 3 | LncRNAs in cancer phenotypes.

Neurodegenerative diseases

LncRNAs are widely expressed in the nervous system and play important roles in CNS development, neuronal differentiation and functions. The involvement of lncRNAs in neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD), are becoming increasingly evident [10]. For example, BACE1-AS, an antisense lncRNA of Beta-secretase 1 (BACE1) gene, is highly expressed in AD patients. It increases BACE1 mRNA stability and hence the Aβ42 amyloid protein production, leading to the disease progression.

It is now widely understood that lncRNAs could identify cellular pathologies, provide diagnostic/prognostic value, or even inform therapeutic options. Spatial, temporal, and disease-associated regulation of expression suggests that lncRNA can be powerful and effective biomarkers. The tissue and cell type specificities of lncRNAs are much higher than mRNAs [21]. Numerous studies have demonstrated the biomarker utilities of lncRNAs (Table 1). For example, LncRNA MT LIPCAR is differentially expressed in left ventricular remodeling and heart failure, as validated in 788 patients. High circulating levels of MT LIPCAR independently predicted adverse cardiac remodeling not confounded by other predictive markers of cardiovascular death.